The long-term goal of this project is to develop human neural stem cell (hNSC)-based therapy to treat diseases involving motoneuron damage. The hypotheses of the present proposal are 1) that hNSC-derived cholinergic neurons will survive indefinitely in the cord, 2) that they will mature and integrate into the organization of the cord, and 3) that they will replace motoneurons lost after neonatal axotomy and thus significantly improve function. The Specific Aims are: 1) to determine optimal parameters for transplantation, 2) to determine the survival and maturation of the hNSC-derived transplanted neurons in a model of motoneuron damage and 3) to show in this model of motoneuron damage that hNSC-derived cholinergic motoneurons attach to muscle cells, that they integrate into the synaptic architecture of the cord and that the animals show behavioral improvement as the transplanted neurons mature. The model of motoneuron damage is neonatal sciatic nerve crush, which causes a reproducible loss of approximately 40% of L4-L5 motoneurons. For Aim 1, different numbers of hNSCs labeled by green fluorescent protein (GFP) will be injected in different volumes into normal cords. Optimization criteria are highest numbers of surviving cholinergic hNSC derived cells, determined stereologically. For Aim 2, motoneuron loss after neonatal axotomy and the numbers of new cholinergic motoneurons derived from hNSCs will be determined over time. For Aim 3, connections of the new motoneurons to muscles will be demonstrated by retrograde tracing, the integration of these new cholinergic neurons into the synaptic architecture of the cord will be shown by light and electron microscopic determination of the numbers and types of synapses that form on the transplanted motoneurons; and behavioral improvements will be assayed by a reliable, efficient gait analysis. These data will provide a beginning assessment of the use of stem cell therapy for axotomy derived motoneuron loss and hopefully give insights into treatment of diseases such as amyotrophic lateral sclerosis and poliomyelitis.